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Abstract

The design and fabrication of custom-tailored microarrays for use as phantoms in the characterization of hyperspectral imaging systems is described. Corresponding analysis methods for biologically relevant samples are also discussed. An image-based phantom design was used to program a microarrayer robot to print prescribed mixtures of dyes onto microscope slides. The resulting arrays were imaged by a hyperspectral imaging microscope. The shape of the spots results in significant scattering signals, which can be used to test image analysis algorithms. Separation of the scattering signals allowed elucidation of individual dye spectra. In addition, spectral fitting of the absorbance spectra of complex dye mixtures was performed in order to determine local dye concentrations. Such microarray phantoms provide a robust testing platform for comparisons of hyperspectral imaging acquisition and analysis methods.

Figures (5)

(a) A printing design is chosen or created in a program such as Photoshop. Here the U.S. Department of Commerce logo, containing 5 colors, is shown in a reduced image size and consists of five colors. (b) The colors represent printing wells (four of which correspond to four distinct colors, with white indicating that a spot should not be printed at that location). (c) The image and well codes are fed into MATLAB, which generates the appropriate code to drive the SpotBot II (see Media 1), as shown here. (d) The image was printed on Epson glossy photo paper to illustrate the printing procedure by showing the spots macroscopically. (e) A close-up comparison of a region of the inputted image and its corresponding printed image.

Examples of detected images from the hyperspectral data cubes of a printed spot on glass. Signal and dark signal intensities are shown for the glass background datacube (Io), and the sample datacube (I). These intensities are used to calculate the absorbance image A. All images are from the same spatial location on the CCD (i.e., the same x,y coordinates). Ring patterns within the hemispherical PEG-based spot are caused by scattering and refraction of the illumination light.

(a) A pattern designed in Photoshop (shown with identifying notations), was used to generate a test microarray that consists of PEG spots containing varying amounts of three spectrally dissimilar dyes (AR, BBR, EG). (b) This sample was imaged by a hyperspectral microscope (shown here for 695 nm illumination). Scalebar is 200 μm. (c) The absorption spectra of dye spots measured by the hyperspectral microscope are shown before (top) and after (bottom) subtraction of the PEG background.

(a) Spectral data collected from the microarray shown in Fig. 3 are displayed along with fitted spectra, obtained by a linear combination method. (b) The same process, applied to two dyes with similar absorption profiles. The number after the name of a dye indicates the percentage of the dye relative to the maximum printed solution concentration.

Tables (1)

Table 1 Expected and calculated concentrations from linear mixing analysis of two dyes: AR and NC. Concentrations are relative to the maximum printed solution concentration. Standard deviation is calculated from the analysis of three different microarrays on the same slide.

Metrics

Table 1

Expected and calculated concentrations from linear mixing analysis of two dyes: AR and NC. Concentrations are relative to the maximum printed solution concentration. Standard deviation is calculated from the analysis of three different microarrays on the same slide.